This study aimed to compare the changes in the axial length (AL) in myopic children that wear centered and decentered orthokeratology (Ortho-K).This retrospective study included 217 subjects who were treated with an Ortho-K lens for >12 months. The subjects were divided into three groups based on the magnitude of the Ortho-K lens treatment zone decentration: mildly, moderately, and severely decentered groups. Distance and direction of treatment zone decentration were calculated using software that was developed in-house. The AL changes in different groups were compared.Based on the distance of the treatment zone decentration, 65 children (65 eyes) were included in the mildly decentered group, 114 children (114 eyes) in the moderately decentered group, and 38 children (38 eyes) in the severely decentered group. The mean decentration distance in the three groups was 0.35 ± 0.11 mm, 0.71 ± 0.13 mm, and 1.21 ± 0.22 mm, respectively. The mean AL increase in the three groups after 12 months of Ortho-K lens wear was 0.24 ± 0.21 mm, 0.23 ± 0.18 mm, and 0.19 ± 0.20 mm, respectively. There were no significant differences in AL changes among the three groups.Ortho-K lens decentration is common in clinical practice. The AL change after Ortho-K lens wear was not significantly different in subjects with different magnitudes of Ortho-K lens decentration. Fitting the Ortho-K lens in the properly centered zone is recommended to ensure the safety of Ortho-K lens wear and to maintain visual quality. Myopia (which is known as near-sightedness) is an abnormal eye condition, in which images from distant objects are focused in front of the retina, which results in blurred vision. Myopia is a major cause of visual impairment, and its prevalence is increasing globally. It has been estimated that the population of myopic patients will rise to approximately 5 billion globally by 2050 . Myopia Orthokeratology (Ortho-K) is a popular, non-surgical method that has been widely used to correct myopia and can control myopia progression in adolescents –6. An OrThe ideal shape of the cornea after Ortho-K lens wear includes a centrally flattened zone and a bulls-eye pattern on the corneal tangential difference map. However, the center of the Ortho-K lens treatment zone is often inconsistent with the center of the pupil in Ortho-K lens wearers. This Ortho-K lens decentration might be caused by multiple factors, which include eyelid tension, corneal astigmatism, or Ortho-K lens movements . Ortho-KControversial conclusions remain in the literature on the effect of Ortho-K lens decentration on the progression of myopia. For example, Wang et al.  found thThis study aims to analyze the effect of centered and decentered Ortho-K lens wear on myopia. In addition, the AL changes after Ortho-K lens wear for>12 months were compared between groups with different magnitudes of Ortho-K lens treatment zone decentration.This retrospective study included 217 subjects who wore Ortho-K lens for >12 months in the myopic control outpatient clinic of Beijing Tongren Hospital  from January 2017 to December 2019. This study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Beijing Tongren Hospital. Written informed consent form was obtained from all subjects and their guardians.The inclusion criteria were: (1) a spher ical equivalent refractive error (SER) of <–6.00 diopter sphere (DS); and (2) a best-corrected visual acuity (BCVA) of 20/20 or better after the removal of the lens. To eliminate the between-eye correlation, the right eyes of the subjects were included in this study. Subjects who had strabismus, systemic diseases, or complications associated with Ortho-K lens wear, such as glare or corneal epithelial defect, were excluded from this study.All the subjects underwent comprehensive baseline ocular examinations, which included slit-lamp examination, uncorrected visual acuity measurement, BCVA obtained with manifest refraction, fundus examination, and AL measurement. Cycloplegic refraction was performed at the first visit, and cycloplegia was achieved by applying four drops of compound tropicamide eye drops  with 5 m intervals. Then, 10 minutes after the application of the fourth drop, autorefraction was performed three times  and the mean value was calculated. AL was measured using a noncontact partial coherent interferometry . Corneal Topographer  was used to obtain the corneal parameters by the same professional technician.After Ortho-K lens fitting, all subjects were advised to wear their lenses every night for ≥8 consecutive h. Examinations were performed 1 day, 1 week, 1 month, and every 3 months after Ortho-K lens wear. At each visit, all subjects received a detailed slit-lamp examination, BCVA measurement, and corneal topography. AL measurements were carried out every 6 months.A Corneal Topographer (Medmont E300) was used to collect the corneal surface data from the subjects and the tangential power map of corneal topography after 1 month of Ortho-K lens wear was used for analysis. An in-house developed computer program (Python) was used to measure the distance of the Ortho-K lens decentration. The data on the tangential radius of curvature and its coordinates were obtained from a Medmont Topographer. Then, the data on the tangential radius of curvature were converted into data on the tangential diopter, and the noise points generated during the acquisition process were removed by mean filtering. The sets of all maximum points in each direction (300 directions) of the tangential diopter were clustered with the K-means algorithm, and all the points in the decentered zone were identified. The data points obtained with the K-means algorithm were fitted to obtain a fitted ellipse. The distance between the center of the ellipse and the center of the pupil was defined as the decentration distance, and the severity of decentration was classified based on the decentration distance. Figure The definition of the magnitude of the treatment zone decentration proposed by Tsai and Lin was used in this study . The subp<0.05.Continuous variables were presented as mean ± standard deviation (interquartile range (IQR) where appropriate. Categorical variables were presented as numbers and percentages (%). The Shapiro-Wilk test was performed to examine if the continuous variables were normally distributed. Group differences in baseline characteristics and ALs were analyzed using the Kruskal–Wallis H test or one-way analysis of variance (ANOVA). To compare the changes in AL in the three groups, covariance analysis with the covariates adjusted was conducted. Multiple linear regression was used to analyze the associations between potential variables and AL. All statistical analyses were performed using SPSS software  with the significance level established at two-sided Throughout this study, all subjects maintained a visual acuity >20/20 or better. After 1-month Ortho-K lens wear, the treatment zone decentration distance in the mildly, moderately, and severely decentered groups was 0.35 ± 0.11 mm (0.01–0.49 mm), 0.71 ± 0.13 mm (0.50–0.97 mm), and 1.21 ± 0.22 mm (1.00–1.81 mm), respectively. For the horizontal displacement, temporal decentration was observed in 173 eyes (79.7%). For the vertical displacement, inferior decentration was observed in 123 eyes (56.7%). For the overall displacement, inferotemporal decentration was the most commonly observed decentration and occurred in 97 eyes (44.7%). Figure p>0.05, Table In total, there were 65 subjects  in the mildly decentered group, with a mean age of 12.49 ± 1.75 years (10–19 years), 114 subjects  in the moderately decentered group, with a mean age of 12.45 ± 1.58 years (10–17 years), and 38 subjects  in the severely decentered group, with a mean age of 12.26 ± 2.10 years (9–19 years). No statistical difference was found in age, gender, baseline AL, spherical refractive error, astigmatism, equivalent e-value, flat K, steep K, and deviated angle . On average, the overall AL change decreased by 0.037 mm per year. However, there was no significant difference in the age-associated AL change between the moderately decentered group and the severely decentered group , and the average AL change decreased by 0.037 mm per year. This result agreed with a previous study that was conducted in Singapore, which reported that the annual AL increase in myopia decreased with age [In this study, the multi-factor linear regression results showed that age was an influential factor for AL elongation  and insufficient sample size. In future studies, to explore the relationship between peripheral defocus and myopia control effect, a quantitative algorithm should be established to calculate the peripheral defocus after Ortho-K wear.No statistical difference in the effect of myopia control was found among the mildly, moderately, and severely decentered groups. To maintain visual quality and ensure the safety of Ortho-K lens wear, fitting an Ortho-K lens in the central position is recommended.


While there was no significant difference in %CEC, caspase-3/7 activity or MCP-1 secretion between the MACE(−) and MACE(+) groups, total CEC and HDL cholesterol (HDL-C) in the MACE(+) group were significantly lower than those in the MACE(−) group. Total CEC was correlated with HDL-C. A receiver-operating characteristic curve analysis showed that there was no significant difference between the areas under the curves for total CEC and HDL-C. In conclusion, total CEC in addition to HDL-C, but not %CEC, was associated with the presence of MACE. On the other hand, HDL functionality with regard to anti-inflammatory and anti-apoptosis effects was not associated with MACE.The present study aimed to investigate the associations between high-density lipoprotein (HDL) functionality and major adverse cardiovascular events (MACE) in patients who have undergone coronary computed tomography angiography (CCTA). We performed a prospective cohort study and enrolled 151 patients who underwent CCTA and had a follow-up of up to 5 years. We measured cholesterol efflux capacity (CEC), caspase-3/7 activity and monocyte chemoattractant protein-1 (MCP-1) secretion as bioassays of HDL functionality. The patients were divided into MACE(−) ( In patients who are treated for atherosclerotic cardiovascular disease (ASCVD), there is a possibility of some residual risks even when the low-density lipoprotein cholesterol (LDL-C) level has been significantly reduced ,2. Such Coronary computed tomography angiography (CCTA) has become more widely available in many general hospitals and enables the accurate non-invasive assessment of coronary artery stenosis for screening of coronary artery disease (CAD). In our previous cross-sectional study, high levels of HDL-C at the time of CCTA were associated with a reduced presence and severity of CAD . In addin = 8) and an insufficient volume of blood samples for further analysis of HDL functionality (n = 45), and finally analyzed HDL functionality in 151 patients.In our previous study, 204 consecutive subjects who underwent CCTA for screening of CAD and were either clinically suspected to have CAD or had at least one cardiovascular risk factor were enrolled, and CEC was measured . In thisn = 13) and without ((−) group, n = 138) a major adverse cardiovascular event (MACE), where MACE was defined as all-cause deaths, acute myocardial infarction, coronary revascularization and ischemic stroke as a composite primary endpoint. When the patients had significant coronary stenosis as assessed by CCTA and received coronary intervention immediately after CCTA, the intervention was not included in MACE as coronary revascularization. We measured HDL functionality including CEC, caspase 3/7 activity associated with apoptosis and the secretion of monocyte chemotactic protein-1 (MCP-1) associated with inflammation. The protocol in this study was approved by the ethics committee of the Fukuoka University Hospital (# 09-10-02). All subjects gave their written informed consent to participate.We followed the patients for up to 5 years  and divided them into those with ((+) group, We assessed coronary stenosis using CCTA ,14. All 2). BP was determined as the mean of two measurements obtained in an office setting by the conventional cuff method using a mercury sphygmomanometer after at least 5 min of rest. The presence of dyslipidemia (DL), hypertension (HTN), diabetes mellitus (DM) and use of medication were obtained from medical records. Patients who had SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg at present or who were taking antihypertensive treatment were considered to have HTN. Patients with LDL-C ≥ 140 mg/dL, TG ≥ 150 mg/dL and/or HDL-C < 40 mg/dL or who were taking lipid-lowering treatment were considered to have DL. Patients with FBG ≥ 126 mg/dL, HbA1c ≥6.5% or who were receiving a glucose-lowering drug were considered to have DM. We calculated the estimated glomerular filtration rate (eGFR) from the data of serum creatinine, age, body size and gender. We defined CKD as when eGFR was less than 60 mL per minute per 1.73 m2 body surface area.Age, gender, body mass index (BMI), systolic blood pressure (SBP), diastolic BP (DBP), smoking status (current vs. nonsmoker), family history , angina pectoris or sudden death) and chronic kidney disease (CKD) were collected as risk factors for CAD. Data of serum levels of LDL-C, HDL-C, triglycerides (TG), hemoglobin A1c (HbA1c) and fasting blood glucose (FBG) were also collected. LVEF was assessed by transthoracic echocardiography. BMI was calculated as weight (kg)/height  was added for 4 h. Radiolabeled cholesterol counts were analyzed for the cell compartment and media. Percentage (%) of CEC was calculated as follows: ) × 100-CEC in sample-free medium. Total CEC was also calculated as the percentage of cholesterol efflux capacity/100 × HDL-C level.We examined HDL CEC with an ex vivo system that used J774 macrophages and HDL isolated from plasma of the study patients by ultracentrifugation . BrieflyWe evaluated the HDL-induced secretion of MCP-1 with an ex vivo system using human coronary endothelial cells   and apo-®, CRL-1446T, Manassas, VA, USA) and apo-B-depleted plasma from the study participants as samples. We used cardiomyoblasts to analyze anti-apoptosis by HDL because HDL may prevent the progression of cardiac dysfunction related to apoptosis. H9C2 cells were cultured and grown in media. In the experiments, H9C2 cells were grown under serum-free conditions for 2 h. After 2 h, the cells were incubated with 5 μg/mL of samples for an additional 6 h. The caspase 3/7 activities in the H9C2 cells were measured by the Caspase-Glo® 3/7Assay System . Relative caspase 3/7 activity in each sample was calculated by the ratio of the activity in each sample to the activity in standard HDL. Relative total caspase 3/7 activity was also calculated as the relative caspase 3/7 activity/100 × HDL-C level.We analyzed the HDL-suppressed caspase 3/7 activity with an ex vivo system that used the H9C2 cell line of embryonic rat cardiomyoblasts  curve analysis was used to determine the cut-off of the total CEC or HDL-C to distinguish between with and without MACE at the highest possible sensitivity and specificity levels. Area under the curve (AUC) values were compared between total CEC and HDL-C by a Chi-square analysis. A value of p < 0.05 was considered significant.The statistical analysis was performed using IBM SPSS statistics software, version 22  and EZR, which is used in R . More precisely, it is a modified version of R Commander designed to add statistical functions frequently used in biostatistics . Continu2 in all patients. The frequencies of HTN, DL and DM in all patients were 78%, 71% and 23%, respectively. The MACE(+) group showed a higher level of %smoking and a lower level of HDL-C than the MACE(−) group. There were no significant differences in other factors, including %CAD, the number of VD, Gensini score, left ventricular ejection fraction (LVEF), %CKD, eGFR and medications between the MACE(+) and MACE(−) groups.p = 0.030) and HDL-C levels (p = 0.014) than the MACE(−) group, while there was no difference in %CEC between the groups.As shown in p = 0.819), relative total caspase 3/7 activity (p = 0.745), the relative secretion of MCP-1 (p = 0.235) or relative total secretion of MCP-1 (p = 0.307) between the groups.r = 0.793, p < 0.001), whereas %CEC showed no correlation   A,B.p = 0.656), which indicated that these two factors contributed to MACE to a similar extent.A ROC curve analysis showed that the AUC for total CEC and HDL-C were 0.682 and 0.696, respectively, in all patients A,B. The In this study, we hypothesized that HDL functionality, CEC in particular, was associated with MACE. The main finding was that total CEC and HDL-C in the MACE(+) group were significantly lower than those in the MACE(−) group. In addition, total CEC was correlated with HDL-C. These two factors contributed to MACE to a similar extent. On the other hand, HDL functionality with regard to anti-inflammatory and anti-apoptosis effects was not associated with MACE.We showed that total CEC and HDL-C in the MACE(+) group were significantly lower than those in the MACE(−) group. We measured %CEC using isolated HDL by ultracentrifugation to eliminate the effects of other lipoproteins as much as possible, since several studies have reported that other lipoproteins might influence cholesterol efflux capacity ,18. By tHDL functionality with regard to anti-inflammation and anti-apoptosis was not associated with MACE in this study. Many studies have shown that vascular inflammation is associated with adverse events and C-reactive protein is a critical biomarker of CVD ,28,29,30This study has several important limitations. First, although the sample size was relatively small, which limited our ability to determine significance, such as in the ROC analysis, including the cut-off levels of total CEC and HDL-C, which may be affected by gender-specific differences, we found that total CEC clearly had a significant correlation with MACE. Second, the population was only selected from Japan and the findings may not be applicable to other populations. Third, the CEC assay itself has several limitations because cell-based assays are labor-intensive. We did not analyze the anti-oxidative function of HDL. Fourth, we divided patients according to the presence of MACE and the duration of follow-up was only up to five years. A large-scale survey with a longer follow-up and further analysis will be needed.Total CEC was correlated with HDL-C. Total CEC in addition to HDL-C, but not %CEC, was associated with the presence of MACE. These two factors contributed to MACE to a similar extent. On the other hand, HDL functionality with regard to anti-inflammatory and anti-apoptosis effects was not associated with MACE.


As a result of laying up ABS sheet on C-PLA sheet, the toughness of resulting structure increased greatly (280 to 365%) as compared to the equivalent C-PLA structure, as expected. Two different types of distinct failures were observed during impact testing. In type A, both laminates fractured simultaneously without any delamination as a hammer hit the sample. In type B, the failure initiated with fracturing of C-PLA sheet followed by interfacial delamination at the boundary walls. The SEM analysis of fractured surfaces revealed two types of pores in the C-PLA lamina, while only one type in the ABS lamina. Further, there was no interlayer cracking in the C-PLA lamina contrary to the ABS lamina, thereby indicating greater interlayer adhesion in the C-PLA lamina.Nowadays, the components of carbon fiber-reinforced polymer composites  are directly produced with 3D printing technology, especially Fused Filament Fabrication (FFF). However, such components suffer from poor toughness. The main aim of this research is to overcome this drawback by introducing an idea of laying down a high toughness material on the 3D-printed carbon fiber-reinforced polymer composite sheet, thereby making a hybrid composite of laminar structure. To ascertain this idea, in the present study, a carbon-reinforced Polylactic Acid (C-PLA) composite sheet was initially 3D printed through FFF technology, which was then laid upon with the Acrylonitrile Butadiene Styrene (ABS), named as C-PLA/ABS hybrid laminar composite, in an attempt to increase its impact toughness. The hybrid composite was fabricated by varying different 3D printing parameters and was then subjected to impact testing. The results revealed that toughness increased by employing higher layer thickness and clad ratio, while it decreased by increasing the fill density, but remained unaffected due to any change in the raster angle. The highest impact toughness (23,465.6 kJ/m Laminar composites are made up of a series of lamina. By the choice of laminar architecture  and constituent materials, laminar composites can be engineered to prepare a structure with required properties . Upon loHand lay-up, filament winding, and compression molding are the common conventional methods to fabricate laminar composites ,4. BladeOn the other hand, Fused Filament Fabrication (FFF), an innovative process, does not suffer from the aforementioned issues because it offers flexible and dye-free fabrication ,9. It isAttempts have been made to enhance the mechanical properties of 3D-printed components with the addition of particles and fibers. This technique has been employed in the polymer industries to increase the structural strength of the 3D-printed components . Zhong eCarbon fiber-reinforced polymer composites are gaining popularity owing to their extensive high-end applications. Nowadays, it has become a common practice to build carbon fiber-based polymer components using the FFF technique. Although such composite components exhibit high strength, these suffer from poor toughness. Various studies have been carried out to investigate impact toughness of FFF-printed materials. Vidakis et al.  performeThe FFF-built carbon fiber-based polymeric composites reported so far have been mainly fabricated as monolithic/single sheets ,30. As cFrom the above literature analysis, it is concluded that impact toughness of the FFF-built laminar composites is not reported in the literature. Further, the idea to enhance the toughness of carbon fiber-based polymer composites by overlaying it with a tougher polymer is yet unexplored. The present study is aimed at developing a laminar composite (composed of two laminates) and characterizing its impact toughness in order to examine usefulness of the proposed idea. The laminates are fabricated in two filament materials, namely, Carbon-fiber reinforced Polylactic Acid (C-PLA) and Acrylonitrile Butadiene Styrene (ABS), thus resulting in a laminar hybrid composite. The reason of choosing C-PLA is that it is a carbon-filled composite filament with a high strength and stiffness available commercially. The ABS was selected as it is a polymer alloy with a reasonable mix of various mechanical properties and high toughness, thereby supplementing low toughness of C-PLA lamina. The hybrid composite is made by varying boundary wall configuration and printing parameters, namely, layer thickness (LT), raster angle (RA), fill density (FD), and clad ratio (CR). The toughness of the printed composite samples is determined by performing an impact test. The results confirm that superimposing ABS on C-PLA lamina can substantially raise the toughness of C-PLA sheet. Moreover, an optimum set of parameters yielding high toughness is also proposed.Two types of filament materials were employed, namely, ABS and C-PLA. True white ABS filament is an alloy of three polymers, namely, acrylonitrile, butadiene, and styrene. C-PLA is a composite filament prepared by mixing 4043D PLA resin with chopped short carbon fibers (15% wt) . Both of these filaments had a diameter of 1.75 mm (+0.5 mm). Their properties are listed in The 3D printing temperatures and bed temperatures for ABS are 220–230 °C and 80–90 °C, respectively. For C-PLA, these temperatures are 190–210 °C and 50 °C, respectively . C-PLA iThe impact test geometry, according to ISO 179-1 standard, employed in this study is shown in The impact samples can be 3D printed either with boundary wall or without boundary wall. Therefore, some preliminary tests were performed to explore the effect of boundary wall. Individual specimens of ABS, C-PLA and ABS/C-PLA hybrid composite were made using the FD of 40% and 100%, feed of 20mm/s and LT of 0.5 mm. 1 = Initial height of hammer, h2 = Final height of hammer after striking, E = Impact energy to fracture the specimen, and A = cross-sectional area.Impact energy can be found by following equations: To investigate the effect of 3D printing parameters on the impact toughness of the hybrid laminar composite, four different 3D printing parameters, i.e., LT, FD, RA, and CR were varied: see definitions in X = No. of levelssn = No. of parametersA comprehensive test plan was prepared using Design Expert (DX-10) software. Full factorial design was adopted for this experimentation as it explores many factors, setting each factor to only two levels. Full factorials design is useful for the estimation of individual and interactive effects. The mathematical form of design is shown below:The response is dependent on various independent variable inputs. By using the minimum and maximum values of the 3D printing parameters, the test plan of sixteen test runs is shown in −2 was created to remove residual gases for reaction. Current and voltage of 10–15 mA and 1–1.4 KV were maintained, respectively. Conductive layer of copper was sprayed on the fractured surfaces of polymers.In order to analyze the behavior of fractured surfaces, PHILIPS XL-30 with tungsten filament SEM was employed. The surface of polymer sample should be conductive to perform SEM. For this purpose, sputtering was done on the samples. In sputtering, vacuum pressure of 102 for test No. 3 against the following 3D printing parameters: LT = 0.5mm, RA = 45°, CR = 1 and FD = 40%. While the lowest value of impact toughness was 7672.8 kJ/m2 for test No. 14 against the following 3D printing parameters: LT = 0.1 mm, RA = 0°, CR = 0.5, and FD = 100%.The percentage improvement in impact toughness due to laying ABS on C-PLA sheet is presented in p-value ≤ 0.05 (95% confidence level) was regarded as significant. This can be noticed from p-value < 0.0001 is the most significant 3D printing parameter.Analysis of Variance (ANOVA) was performed on the results in order to identify the significant parameters affecting the impact toughness of the composite. A parameter with Contrary to the effects of LT and CR, An empirical model was developed to correlate a response with the complete range of conditions of the entire design. For this purpose, the regression analysis was done using DX-10 software. The following empirical model was yielded for the only response, i.e., impact toughness:Experiments were performed to validate the model, as shown in i = desirability of individual response, and ri = weightage of each response.The desirability function as described by Costa  was usedAs studied above, the impact toughness of the specimen was affected by the 3D printing parameters. Optimum conditions were developed to attain appropriate impact toughness. Optimization was done with an iterative manner and 100 solutions were achieved. 2, respectively, with a difference of 4.7%. It can be observed that optimized results are in agreement with the experimental results. For optimization in this study, LT of 0.5 mm, CR of 1, FD of 40%, and RA of 45° were determined to be the best 3D printing parameters. To verify these 3D printing conditions, a sample was 3D printed and tested. The predicted and actual strength were 22,361.7 and 23,465.6 kJ/mAs shown in The type B failure differed from type A in a sense that it included delamination of ABS boundary wall besides fracturing of remainder composite b. This kFurther, it was observed that the C-PLA lamina of the composite always fractured in a brittle manner making an angle of 90° with the loading axis for both of 0°/90° and 45°/−45° rasters c. The frBoundary wall is found to increase the impact toughness of FFF-built samples. An increase of 60% was obtained due to making boundary wall around the sample.The strategy of coupling ABS lamina with the C-PLA lamina substantially increases the impact toughness (280 to 365%) as compared to that of the equivalent C-PLA lamina.2 in this study. The optimum conditions sought for maximizing the impact toughness suggest that greater layer thickness (0.5 mm), lower fill density (40%), and higher clad ratio (1) are conducive.A change in the 3D printing parameters affects the impact toughness of the hybrid laminar composite, except raster angle appears to be insignificant. The impact toughness is found to range from 7672.9 to 23,465.6 kJ/mTwo different types of failures are observed when the hybrid laminar composite is subjected to impact load. In the type A failure, both laminates fractured simultaneously without any delamination as hammer hit the sample. The type B failure differed from type A in a sense that it included delamination of ABS boundary wall besides fracturing of the remainder composite.Voids in the fractured surface of C-PLA lamina were observed due to absence of material and breaking of fibers, while voids in the ABS fractured surface were noticed only due to absence of material. Further, interlayer cracks were observed in the ABS fractured surface near the pores suggesting poor interlayer adhesion. While, no such crack was observed in the C-PLA fractured surface, indicating higher interlayer adhesion.In this study, ABS polymer was laid upon the 3D-printed C-PLA lamina using FFF technology with an objective to improve its impact toughness. This printing configuration led to the fabrication of a hybrid laminar composite. The composite was produced by varying FFF conditions. Impact tests were performed to find out the impact toughness of the fabricated samples. Several important conclusions were drawn from this study as outlined below:


Hypertensive nephropathy is a chronic kidney disease caused by hypertension. Eicosapentaenoic acid (EPA) has been reported to possess an antihypertensive effect, and our previous study suggested that EPA-enriched phospholipid (EPA-PL) had more significant bioactivities compared with traditional EPA. However, the effect of dietary EPA-PL on hypertensive nephropathy has not been studied. The current study was designed to examine the protection of EPA-PL against kidney damage in spontaneously hypertensive rats (SHRs). Treatment with EPA-PL for three weeks significantly reduced blood pressure through regulating the renin–angiotensin system in SHRs. Moreover, dietary EPA-PL distinctly alleviated kidney dysfunction in SHRs, evidenced by reduced plasma creatinine, blood urea nitrogen, and 24 h proteinuria. Histology results revealed that treatment of SHRs with EPA-PL alleviated renal injury and reduced tubulointerstitial fibrosis. Further mechanistic studies indicated that dietary EPA-PL remarkably inhibited the activation of TGF-β and Smad 3, elevated the phosphorylation level of PI3K/AKT, suppressed the activation of NF-κB, reduced the expression of pro-inflammatory cytokines, including IL-1β and IL-6, and repressed the oxidative stress and the mitochondria-mediated apoptotic signaling pathway in the kidney. These results indicate that EPA-PL has potential value in the prevention and alleviation of hypertensive nephropathy. Hypertension is a common disease that seriously endangers public health . Hyperte−/− mice [Eicosapentaenoic acid (EPA), as an omega-3 long-chain polyunsaturated fatty acid (ω-3 PUFA), is widely distributed in the body of multifarious marine products. Notably, EPA exhibits many important physiological activities, which has been considered by investigators to be beneficial in the prevention or treatment of several diseases, such as alleviating cardiovascular disease, improving cognitive function, and lowering blood pressure ,10,11. E−/− mice . Our pre−/− mice . Moreove−/− mice . Further−/− mice ,9,11. HoIn the present study, a spontaneous hypertensive rat (SHR) model was used to determine the effects of EPA-PL in attenuating elevated systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean artery pressure (MAP) by the renin–angiotensin system. Meanwhile, we evaluated the effect of EPA-PL on renal interstitial fibrosis in rats with hypertensive nephropathy through analyzing the transforming growth factor-β (TGF-β)/Smad 2/3-meditated tubulointerstitial fibrosis, the PI3K/Akt/NF-κB signaling pathways, oxidative stress, and the mitochondria-mediated apoptosis signaling pathway.p < 0.05), and the moderate dosage of EPA-PL appeared to decrease the SBP; however, there was no significant difference between the Moderate EPA-PL and M groups. The low dosage of EPA-PL failed to lower the level of SBP in SHRs (p > 0.05). Likewise, the high dosage of EPA-PL distinctly decreased the DBP compared with the model group (p < 0.05), and the moderate and low dosage of EPA-PL were not able to reduce the level of DBP (p > 0.05). MAP is defined as the average arterial blood pressure during a single cardiac cycle, which reflects the hemodynamic perfusion pressure of the vital organs. In the present study, the MAP in the High EPA-PL group was significantly reduced by 16 mmHg relative to the model group (p < 0.05). Dietary moderate and low dosage of EPA-PL had no effect on the decrease in MAP in SHRs (p > 0.05). The short-term plot experiment showed that the high dosage of EPA-PL had a positive antihypertensive effect in SHRs.We firstly analyzed the change in blood pressure after single oral administration with different doses of EPA-PL, as shown in p > 0.05, p < 0.05). After one week of dietary intervention, EPA-PL failed to reduce the SBP in SHRs, interestingly, there was a lower level of SBP in the EPA-PL group after intervention for two weeks (p < 0.05), which was similar to the effect of the intervention for three weeks. Likewise, the level of DBP in EPA-PL group was remarkably reduced with the prolonging of time, especially after two weeks (p < 0.05). Moreover, dietary EPA-PL could distinctly reduce the MAP level after treatment with EPA-PL for two weeks. This indicates that the supplementation with EPA-PL has obvious effects on the prevention and treatment of hypertension. Moreover, the heart index and kidney index were significantly elevated in the model group compared to those in the control group, and there was a lower level of heart and kidney index in the EPA-PL group ) had a notably lowering effect on Ang II in the plasma of Chinese hypertensive patients over a treatment period of 90 days [The renin–angiotensin system performs a vitally important role in maintaining the balance of blood pressure in the body. However, when the renin–angiotensin system (RAS) is pathologically activated, it could cause excessive vasoconstriction, proliferation and hypertrophy of vascular smooth muscle and myocardium, and even fibrosis . Renin a 90 days .p < 0.05), and the EPA-PL supplementation rapidly raised the expression level of ACE2 (p < 0.05). Both ACE and AT1 protein expression levels in the model group were significantly raised (p < 0.05), and the intervention of EPA-PL inhibited the protein expression levels of ACE and AT1 (p < 0.05). Moreover, dietary EPA-PL significantly raised the expression level of MAS compared with the model group (p < 0.05). Ulu et al. showed that the intake of DHA could increase the mRNA expression of ACE2 and AT1 levels in the kidney of mice with angiotensin II-dependent hypertension [Renin, as an angiotensinogenase, is secreted by the kidneys that participate in the renin–angiotensin system; therefore, the physiological function of the kidney is closely related to the regulation of blood pressure . In the rtension . Accordip < 0.05). These changes were consistent with previous research results [p < 0.05). Toroudi et al. demonstrated that the intervention of DHA + EPA for 14 days at a dose of 200 mg/kg body weight can clearly reduce the BUN and Cr contents in the serum of rats with kidney disease caused by ischemia–reperfusion [The BUN, Cr, and 24 h proteinuria are the main indexes of kidney function biomarkers in clinical diagnosis. We next measured the contents of BUN and Cr in the serum, as well as the 24 h proteinuria of the SHRs A–C. Ther results . This inerfusion . Our preerfusion .l-α-phosphatidylcholine for 30 days at a dose of 100 mg/kg body weight alleviated the kidney pathological injury of HgCl2-treated rats [The H&E staining and Masson staining methods were used to analyze the pathological changes in the kidney for renal function assessment in clinical practices . The patted rats . Likewisted rats . The abop < 0.05). Dietary EPA-PL clearly suppressed the expression of the Smad 3 protein (p< 0.05); however, there was no obvious difference in the protein expression of Smad 2 between the model and EPA-PL groups (p > 0.05). Moreover, there was a high expression of the TGF-β protein in the kidney in the model group . Some reports have suggested that the pre-treatment of EPA significantly inhibited the expression of Smad 3 and TGF-β in renal epithelial cells (HK-2 cells) [Emerging evidence indicates that renal interstitial fibrosis is a typical pathological feature in the development of hypertension-induced kidney damage . In addi2 cells) . Cao et 2 cells) . Additio2 cells) . Our curp < 0.05); furthermore, there was a higher level of p-PI3K and p-Akt expression after treatment with EPA-PL than in the model group ; however, the expression level of Akt was not distinctively changed in the SHRs (p > 0.05). Moreover, the ratios of p-PI3K/PI3K and p-Akt/Akt in the EPA-PL group were drastically increased compared with the model group (p < 0.05). Furthermore, levels of both NF-κB p65 and p-NF-κB p65 were drastically reduced in the EPA-PL group compared with the model group (p < 0.05). Some researchers have reported that n-3 PUFA mediates a protective effect against LPS-induced PC12 cell injury by activating the PI3K/Akt pathway [Accumulating evidence has demonstrated that the PI3K/Akt/NF-κB signaling pathway is closely related to fibrosis, which might be involved in the course of the disease, and previous studies have also found that TGF-β can regulate the phosphorylation of the PI3K protein . Moreove pathway . Additio pathway .p < 0.05), and dietary EPA-PL clearly reduced the expression levels of IL-1β and IL-6 proteins (p < 0.05). TNF-α is a strong pro-inflammatory cytokine that plays an important role in the immune system during inflammation. An increasing number of studies have shown that the NLRP3–ASC–caspase-1 inflammasome is a novel innate immune sensor and pro-inflammatory cytokine, and the NLRP3 inflammasome-mediated caspase-1 activation is a key factor in the development of kidney diseases. In the current study, there was a higher level of TNF-α in the model group compared with the control group (p < 0.05), and the intervention of EPA-PL failed to inhibit the expression of TNF-α (p> 0.05). Similarly, the supplementation of EPA-PL was not able to reduce the abnormal rise in caspase-1 protein level (p > 0.05). Moreover, the expression of ASC and NLRP3 was significantly increased in the model group (p < 0.05), and the intervention of EPA-PL distinctly inhibited the expression of ASC (p < 0.05); however, there was no significant difference for NLRP3 between the model group and EPA-PL group (p > 0.05). Emerging evidence indicates that EPA possesses high anti-inflammatory activity. Dietary supplementation of EPA at the dose of 400 mg/kg body weight by gavage for 63 days significantly suppressed the increase in IL-1β and TNF-α expression levels in the intestinal epithelium of dextran sulphate sodium (DSS)-induced colitis mice [−/− mice [Studies have reported that the activation of the inflammasome and subsequent release of proinflammatory cytokines are involved in renal interstitial fibrosis . A previtis mice . Moreove−/− mice . In addi−/− mice . Some st−/− mice . Our prep > 0.05). The enzyme activity of T-AOC was significantly reduced in the kidney of the model group compared with the control group, and the EPA-PL supplementation remarkably increased the T-AOC activity (p < 0.05). Similarly, the enzyme activity of GSH was clearly decreased in the kidney of the model group to a greater degree than in the control group (p < 0.05), and dietary EPA-PL sharply elevated the GSH activity in the kidney (p < 0.05). Our findings clarify that EPA-PL can inhibit the oxidative stress in the kidney, and thus protect against renal interstitial fibrosis. In accordance with our findings, some studies have shown that the administration of EPA (300 mg/kg per day for two weeks) clearly suppressed MDA activity and elevated the activity of GSH, in the liver of rats with valproate-induced liver toxicity [Recent evidence suggests that oxidative stress is one of the major contributing factors in the development of hypertension. Indeed, some studies have shown that there is a strong link among oxidative stress, NF-κB-meditated inflammation, and interstitial fibrosis in the kidney . MDA, T-toxicity . Likewistoxicity ; and thetoxicity .p < 0.05), and the increase was clearly reduced after the EPA-PL supplementation (p < 0.05). Interestingly, the expression levels of anti-apoptotic protein Bcl-2 were sharply raised in the model group more than in the control group, which might be related to the role of organism self-protection after kidney injury; in addition, the level of Bcl-2 in the EPA-PL group was higher than the model group (p < 0.05). In the current study, the ratio of Bax/Bcl-2 was significantly increased in the model group relative to the normal group, and EPA-PL administration sharply reduced the ratio of Bax/Bcl-2 when compared with the model group (p < 0.05). The increase in the Bax/Bcl-2 ratio could stimulate the release of cytochrome c (Cyt-C) from the mitochondria to the cytoplasm, and then activate caspase-9. At the same time, it has been reported that the inhibitor of apoptosis proteins (IAPs) are a family of proteins that function as intrinsic regulators of the caspase cascade, and may have a crucial role in the inflammatory response in kidney disease [p < 0.05, p < 0.05). The expression levels of cleaved caspase-3 protein were increased in the SHRs more than in the control group, and EPA-PL significantly inhibited the expression of cleaved caspase-3 in the kidneys of the rats (p < 0.05). Furthermore, the expression of cleaved caspase-9 protein showed a rising tendency in the model group, although the results show no significant difference among all groups (p > 0.05). Emerging evidence indicates that the intervention of EPA (a standard chow supplemented with 5% EPA) for two weeks can clearly inhibit the expression of Cyt-C, cleaved caspase-3, and cleaved caspase-9 proteins in the kidney of mice with diabetic tubular injury [A plethora of studies have suggested that apoptosis plays an important role in the development of kidney injury, and that the activation of the apoptosis signaling pathway can further promote renal interstitial fibrosis ,45. Ther disease . Sebe et disease . In the  disease ,50. Notar injury , as wellr injury .n-3 PUFAs from fish and other seafood is high in certain Mediterranean countries. Dietary Approaches to Stop Hypertension (DASH) is a dietary pattern rich in fruits, fish, vegetables, and low-fat dairy products, and is low in meats and sweets [n-3 PUFAs are likely to be important mediators of the protection provided by traditional Mediterranean diets and DASH due to their anti-inflammatory properties [n-3 PUFAs in the Mediterranean diet or DASH might be active ingredients for the prevention of hypertensive nephropathy.The Mediterranean diet comprises a high intake of fruits, vegetables, olive oil, unrefined grains, legumes, and fish; a moderate intake of nuts and red wine; and a low intake of red meat and refined sugar . Consumpd sweets . Nutritid sweets . Epidemioperties . Our resAt present, there are still inconsistent views on the hypotensive effect of EPA in clinical studies. On the one hand, numerous studies have reported that EPA treatment has a strong antihypertensive effect; on the other hand, some research indicates that EPA intervention has no significant effect on blood pressure ,53. MoreCucumaria frondosa) was purchased from the Nanshan aquatic products market . The enzymatic reagent kit of creatinine (Cr), blood urea nitrogen (BUN), malondialdehyde (MDA), total antioxidant capacity (T-AOC), glutathione (GSH), and glutathione peroxidase (GSH-Px) were obtained from Nanjing Jiancheng Bioengineering Institute . The enzyme-linked immunosorbent assay kits for Angiotensin II (Ang II) and renin were provided by Shanghai Elisa Biotech Co., Ltd. . All primary antibodies, β-actin, and secondary antibodies were obtained from Abcam  and Cell Signaling Technology .Sea cucumber  (11 weeks old) and Sprague Dawley rats  were fed under the conditions of 23 °C ± 2 °C, 12 h light/dark cycle, relative humidity 65% ± 5%. All procedures were approved by the Ethical Committee of the College of Food Science and Engineering, Ocean University of China . Each rat was fed in a separate cage.n = 6 each group) after one week of adaptive feeding. They were designated as: model group (M), low dose of EPA-PL treated group  EPA-PL), medium dose of EPA-PL treated group , and high dose of EPA-PL treated group . The SBP, DBP, and MAP of the rats were evaluated as previously performed after the single administration of EPA-PL [SHRs were randomly divided into four groups : the model group fed with a high-fat diet  and another group fed with a high-fat diet plus 2% EPA-PL (EPA-PL). Additionally, the Sprague Dawley (SD) rats were used as the control group (N). The ingredients in diets are shown in g for 15 min and then stored at −80 °C. The levels of serum renin, Ang II, Cr, and BUN were measured according to the instructions of the corresponding commercial kits. The kidney was homogenized with cold saline and centrifuged at 3000× g for 10 min to obtain supernatant for future determination. The activities of MDA, T-AOC, GSH, and GSH-Px were determined according to the instructions of the commercial kit.Serum was collected from the blood by centrifugation at 4500× The kidney tissue was fixed and dehydrated with 4% paraformaldehyde, then the tissue was embedded in paraffin and eventually stained with hematoxylin and eosin (HE) or Masson trichrome. Histological changes in the kidneys were evaluated and graded according to the method outlined in the previous report . Renal pKidney tissue was homogenized by radio-immunoprecipitation assay (RIPA) buffer according to the methods of published research . The prop < 0.05 was considered statistically significant. The Student’s t-test was used to determine the difference between the control group and model group, and the differences among the SHRs groups  were determined and analyzed by one-way analysis of variance (ANOVA) and Tukey’s post hoc test. Different letters represent significant differences among experimental groups, and means with the same letter are not significantly different (p > 0.05).All data are presented as mean ± standard error of the mean (S.E.M). A value of In summary, the present results indicate that the supplementation of EPA-enriched phospholipids could effectively relieve hypertension by regulating the renin–angiotensin system in spontaneously hypertensive rats. Moreover, dietary EPA-PL could alleviate hypertensive nephropathy by inhibiting renal interstitial fibrosis. Further mechanistic studies showed that EPA-PL ameliorated renal interstitial fibrosis by inhibiting TGF-β/Smad-meditated tubulointerstitial fibrosis, activating the PI3K/Akt/NF-κB signaling pathway, regulating oxidative stress, and inhibiting the mitochondria-mediated apoptosis pathway. These findings indicate that EPA-PL may be used as a functional food ingredient for the prevention of hypertensive nephropathy.


ZW-FA was incubated with endometrial cytology samples obtained from patients who underwent dilation and curettage or total hysterectomy. Diagnostic utility was calculated by applying laser confocal microscope, Image-J and statistical models, such as enumeration, receiver operating characteristic curve, logistic regression, support vector machine and decision tree were used. The purity of ZW-FA was > 95% determined by 1H NMR. ZW-FA had the strongest absorption peak at 633 nm in ultraviolet spectra. Photostability of ZW-FA was over 8 hours. In clinical validation, a total of 92 patients were enrolled. The cut-off value of ZW-FA was 49 in enumeration, which was used to distinguish the type of samples. Indicators about diagnostic utility are as follows: sensitivity 90.77%, specificity 62.96%, false-positive rate 37.04%, false-negative rate 9.23%, positive predictive value 85.51% and negative predictive value 73.91%. The samples processed by ZW-FA did not affect further Hematoxylin-Eosin staining and pathological diagnosis. It was an effective cytologic strategy for in vitro diagnosis of endometrial cancer and precancerous change by using ZW-FA.The lack of cytopathologists delays the advancement of screening for endometrial cancer. It was urgent to develop a new dye for rapid diagnosis. Our study aimed to synthesize a targeted folate receptor-α near-infrared (NIR) fluorescent agent, folic acid-zwitterionic NIR fluorophore (ZW-FA), and explore the feasibility for screening of endometrial cancer and precancerous change. Folic acid was conjugated with zwitterionic NIR fluorophore. The preparation of ZW-FA was validated by http://www.chictr.org.cn/index.aspx, identifier ChiCTR1800020123. Endometrial cancer is the most common malignancy of the female reproductive system in developed countries . It is uNear-infrared (NIR) fluorescence imaging has emerged as a non-invasive and real-time visualisation technique. Compared to conventional fluorescent dyes, NIR dyes show ultralow autofluorescence, providing high signal-to-background ratio images . ZwitterFolate receptor-α (FR-α) is a 38 to 40 kDa molecule with a high affinity for folic acid and its derivatives . Overexpin vitro diagnosis of endometrial cancer and precancerous change was proposed to verify the feasibility.Nevertheless, related agents used in the diagnosis of endometrial cancer have rarely been developed yet. In this study, we conjugated folic acid and ZW800-1 to construct a targeted FR-α near-infrared fluorescent agent folic acid-ZW800-1 (ZW-FA) (patent number: ZL201510104185.5). After completing the synthesis and characterization of ZW-FA, a cytologic test for The synthesis of ZW-FA was composed of eight steps. In this part, 3-methyl-2-butanone and 4-hydrazinobenzenesulfonic acid were purchased from Aladdin, 4-(2-carboxyethyl) phenylboronic acid and 1,1,3,3-tetramethoxypropane were purchased from Alfa Aesar and 3-Bromo-N,N,N-trimethylpropan-1-aminium bromide was purchased from Ark Pharm. All solvents and other reagents were of reagent grade quality and purchased commercially.1H NMR, mass spectrometric, ultraviolet spectra and fluorescence spectra. 1H NMR spectra were recorded on Varian unity INOVA-400 spectrometer at 400, taking TMS as an internal standard. Mass spectrometric detection was performed on a spectrometer . Ultraviolet spectra were recorded using ultraviolet spectrophotometer . Fluorescence spectra were recorded using a luminescence spectrometer . Photostability of ZW-FA (7.14 ug/ml) was evaluated in a variety of biological media, including H2O, phosphate-buffered saline (PBS), serum and blood at 37°C under continuous laser exposure at 650-850 nm .The preparation of ZW-FA was validated by 3 and human breast cancer cell line MDA-MB-231 with overexpression of FR-α were conducted as positive cell lines and obtained from the Cell Bank of the Chinese Academy of Sciences . Human umbilical vein endothelial cell line HUVEC was performed as a negative cell line and obtained from the American type culture collection in the United States. All cell lines were grown in complete medium without folic acid and maintained under humid conditions with 5% CO2 at 37°C.Human ovarian cancer cell line SKOV5/well overnight. Each slide was washed 3 times with PBS, and the cells were fixed with 0.5 ml of 4% paraformaldehyde for 30 minutes and washed with PBS again.All cells were cultured on slides in a 24-well plate at 103 was incubated with human folate receptor alpha-1 (FOLR1) antibody  with 10 µg/ml and Alexa Fluor 488 . Then the cell was incubated with ZW-FA at 600 ug/ml for 1 hour at 37°C, washed again with PBS and incubated with DAPI  for 30 minutes.To verify the targeting of ZW-FA to FR-α, a monoclonal antibody validation experiment was performed. SKOV3 was incubated with ZW-FA at 400 ug/ml, 500 ug/ml and 600 ug/ml for 1 hour at 37°C. MDA-MB-231 and HUVEC were incubated with ZW-FA at 600 ug/ml for 1 hour at 37°C. They were washed again with PBS and incubated with DAPI for 30 minutes.Similarly, in concentration-dependent experiment, SKOVFinally, antifade mounting medium  was used for sealing, and laser confocal microscope  was used to observe the results. The excitation wavelengths of DAPI, human FOLR1 antibody and ZW-FA fluorescence were 408 nm, 488 nm and 633 nm respectively. It’s worth noting that the incubation and afterward steps needed to be protected from light.Endometrial cytological samples of enrolled patients had been collected by Li Brush  from patients who had undergone dilation and curettage (D&C) or total hysterectomy during 15 months (07/2018 to 10/2019) –28. The This study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University (XJTU1AF2017LSK-100) and informed written consent was obtained from all patients before the study.The samples were stained with ZW-FA and Hematoxylin-Eosin (H&E) successively. The slides loaded with endometrial cells were fixed in 95% alcohol for 30 minutes and washed with PBS . We incuAfter data collection, H&E staining and cytopathological diagnosis were performed by two experienced pathologists to verify whether ZW-FA staining affected subsequent H&E staining . For samTo evaluate the diagnostic utility of ZW-FA, postoperative histopathologic results were regarded as the gold standard . The cytThe false-negative and false-positive samples of ZW-FA were subjected to immunocytochemistry (ICC) to identify whether FR-α was expressed by using human folate receptor alpha-1 (FOLR1) antibody  with 20 µg/ml .The staining intensity of FR-α was divided into 0, no staining; 1, weak; 2, moderate and 3, strong, as described by Henry et al. . EvaluatFluorescence images of each sample were collected by laser confocal microscope. Image-J (Image-J 1.52a) was used to analyze all images and obtain the grey values of them. Then statistical prediction models were built to analyze the above grey values. Statistical prediction models included receiver operating characteristic curve (ROC), enumeration (ENUM), logistic regression, support vector machine (SVM) and decision tree. The ROC curve was used to obtain the cut-off value to calculate the diagnostic utility. The diagnostic utility corresponding to each gray values were listed in ENUM method, and the most appropriate cut-off value would be obtained by comparing all the results. Logical regression model was established to predict the positive probability of each gray value, and then the ROC curve was drawn to calculate the diagnostic utility. The SVM model was used to analyze the data from training set to distinguish the positive and negative in the test set. The cut-off value was calculated by using the decision tree model and then the diagnostic utility was calculated.in vitro diagnosis. Indicators were as follows: sensitivity (Se), specificity (Sp), false-positive rate (FPR), false-negative rate (FNR), positive predictive value (PV+) and negative predictive value (PV–). By comparing the results of each model, an optimal model for endometrial cancer screening was obtained.According to the models, all cytological samples were divided into positive and negative. Indicators about diagnostic utility of ZW-FA were calculated through the confusion matrix for evaluating the feasibility in endometrial cytology All of the data were analyzed by SPSS 22.0  and R-program .1H NMR . The fluorescence signal in the green channel was strong, while the fluorescence signal in the red channel was weak . The red channel of FR-α positive cells SKOV3 and MDA-MB-231 showed a strong fluorescence signal  was 0.881 . AccordiIn ENUM, the cut-off value of ZW-FA was 49, which was used to distinguish the type of samples. According to the confusion matrix  and a test set (28 cases). According to the confusion matrix  . The patients/participants provided their written informed consent to participate in this study.QL designed and configured the study. DL conducted the experiments and wrote the paper. XT conducted the experiments and wrote the paper. QW helped to conduct the experiments. LZ helped to conduct the experiments and polish the manuscript. KZ collected the samples. YW collected the samples. XF collected the samples. PY collected the samples. LG collected the samples. YL helped to conduct the experiments. LW collected the samples. LH collected the samples. RA helped to polish the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Major Basic Research Project of Natural Science of Shaanxi Provincial Science and Technology Department , the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University, China , the Key Research and Development Project of Shaanxi Provincial Science and Technology Department , the Shaanxi Provincial Collaborative Technology Innovation Project , and the Medical Research Project of Xi’an Social Development Guidance Plan (2017117SF/YX011-3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Treating intracranial aneurysms with flow-diverting stents sometimes requires deployment of a second device. Herein we quantify the sizing effects of devices in dual-stent treatments upon the final stent microstructure and the post-treatment aneurysmal haemodynamics.Fifteen sidewall ICA aneurysm geometries were included. Using a virtual stenting technique, we implanted either one or two stents for each aneurysm treatment considered, with each stent specified as one of two different sizes, yielding a total of two single-stent and fouir dual-stent treatment scenarios for each aneurysm. Three stent microstructural parameters and nine aneurysmal haemodynamic parameters were quantified and systematically compared across the 90 treatment scenarios.p = 0.001) and 9 ± 12% (p = 0.056), relative to the untreated condition. Sizing effects of the earlier-deployed stent led to largest differences of 6.9% for the final IR reduction and 11.1% for the EL, whereas sizing effects from the later-deployed stent were minor (≤2.1%). The change in stent pore size was the only microstructural parameter demonstrating a strong correlation with the reduction in the post-treatment aneurysmal haemodynamics, in terms of the IR  and pressure drop .Deployment of a second stent further reduced the aneurysmal inflow rate (IR) and energy loss (EL) by, respectively, 14 ± 11% (Size of the earlier-deployed stent has substantial effects on the final haemodynamic outcomes after dual-stent treatment. The average pore size of stent wires at the aneurysm orifice shows promise as a potential index for predicting the efficacy of flow-diversion treatments. Flow-diverting (FD) stent intervention has become a common endovascular therapy, especially for the treatment of fusiform and wide-necked intracranial aneurysms (IAs) . By placComplete aneurysm occlusion is closely associated with sufficient flow diversion produced by the stent wires that partially cover the aneurysm orifice . For thiTreatments with dual stents of different diameters may result in distinct haemodynamic outcomes, due to the variety of wire configurations following multi-stent implantation . HoweverTo fill in the gaps, we quantified the changes in stent morphology and IA haemodynamics in a cohort of 15 patient aneurysms, before and after dual-stent treatment with devices of different sizes, with reference to single-stent treatment, seeking (i) to establish the correlation between the stent microstructural characteristics  and the resulting IA haemodynamics ; and (ii) to examine whether choice of the stents would have determinative effects on the final flow-diversion efficacy after dual-stent implantation.Aneurisk (n = 103) in accordance with the following exclusion criteria: (i) aneurysms located in arteries other than the ICA segment; (ii) aneurysms with draining arteries or blebs developed on the wall; (iii) aneurysms initiated at an arterial bifurcation; (iv) multiple aneurysms developed adjacent to each other; and (v) aneurysms deemed unsuitable to be treated with FD stents in accordance with the Instructions for Use (IFU)   of the PAneurisk repository. Use of the data from the Aneurisk repository had been approved by the ethical committee of the Ca’ Granda Niguarda Hospital, Milano, Italy.We set these criteria to exclude cases where precise segmentation of the vasculature may be difficult; and to include only aneurysms with similar parent-arterial flow conditions for further haemodynamic and statistical analyses. Geometries of the included aneurysms were immediately acquired from the For the included cases, we modeled flow-diversion treatments with the PED. Virtual deployment of the PED was realised through use of a previously reported and validated spring–mass method . , denoted “L.” To model the plausible dual-stent treatments, we deployed FD stents at each of the two sizes, respectively, into each of the earlier-deployed ones, yielding a total of four dual-stent scenarios—“fit into fit (FinF),” “fit into larger (FinL),” “larger into fit (LinF),” and “larger into larger (LinL).”Given the large longitudinal variation of the parent-arterial diameter within each of the cases, a range of stent diameters could be selected based upon the treating clinicians’ judgements. We therefore investigated the single-stent treatment for each aneurysm geometry with two plausible device diameters determined following the IFU : one thaThe local microstructure of stents was examined following the ISO standard for evaluation of cardiovascular implants , throughSp), and pore density (Dp) were calculated as:For each treatment scenario, we first obtained a series of images by projecting the stent wires onto the tangent planes at each “node” of the outer-layer stent . From eaAROI denotes the area of the ROI; iA refers to the area of the ith pore; and n the number of pores within the ROI. Due to the variation in pore size within the neck plane, a weighted mean evaluation was determined for the pore size in order to give more importance to bigger pores, which have a greater influence on the flow resistance. For each of the three parameters, the mean and SD corresponding to the projections of nodes within a reference neck surface (determined on a patient-by-patient basis) were calculated and used for further analysis  at the Institute of Fluid Science, Tohoku University was used, and the average simulation time for each case was around 6 h with 32 cores in parallel.SnappyHexMesh embedded in the open-source CFD library OpenFOAM  was usedIso-velocity surfaces, streamlines, and wall shear stress were first examined for each treatment scenario. To quantify the flow-diversion outcomes, we calculated the reductions in the IR, PD, EL, aVEL, aVOR, aWSS, mVEL, mVOR, and mWSS.t-tests were used to compare the changes in stent microstructure and IA haemodynamics between treatments with a single stent (n = 30) and with dual stents (n = 60). Correlations between changes in the stent microstructure and changes in the IA haemodynamics after dual-stent implantation were assessed by examining the Pearson correlation coefficient, r.Independent two-sided t-tests were performed . For dual-stent treatments: (i) Pearson χ2 tests were used to examine the sizing effects of the earlier-deployed stent; and (ii) paired-sample two-sided t-tests were used to compare the differences between the later-deployed stents of different sizes .To investigate differences in the stent microstructure and IA haemodynamics after treatments with single stents of different sizes, paired-sample two-sided p < 0.05 was considered suggestive of statistical significance, while p < 0.005 was deemed to confirm statistical significance.The statistical analyses were carried out using MedCalc version 19.1 , supplemented with MATLAB R2019b. Throughout this work Fifteen geometries of aneurysms from 15 patients , at mean age 50 years (range: 26–85) met the inclusion criteria. The included IAs had a mean aspect ratio of 1.27 (range: 0.64–1.76), a mean neck width of 5.98 mm (range: 2.61–9.34), and a mean dome-to-neck ratio of 1.27 (range: 0.83–1.77). The mean volumetric flowrate at the ICA inlet was 2.75 ± 1.30 mL/s. See –2, 71 ± 2%, and 0.039 ± 0.01 mm2 . See Across the 15 cases, the average pore density, porosity, and pore size at the aneurysm orifice after single-stent treatment were, respectively, 20.3 ± 5.2 mm0.01 mm2 . After tp = 0.001) and 9 ± 12% (p = 0.056), in contrast to the PD, which was increased by 22 ± 32% (p = 0.070); and (ii) the intra-aneurysmal aVEL, aVOR, and aWSS were each further decreased by around 10% , in contrast to the mVEL, which was increased by 14 ± 50% (p = 0.453).  We observed that deviations in the maximal haemodynamic parameters were much greater than those in the averaged ones, as maximal parameters are sensitive to local vascular geometries, especially after the insertion of a stent.Attributable to the second stent: (i) the IR and EL were further reduced by 14 ± 11% (The greatest and the poorest reductions of the IR were observed in Case 1 (21%) and Case 9 (67%) after deployment of a single stent, and in Case 1 (13%) and Case 6 (49%) after deployment of dual stents , suggesting that the flow diversion achievable depends strongly upon the geometry of the recipient artery and the location of aneurysm occurrence.p < 0.001). However, this did not lead to a statistically significant difference in the post-treatment IA haemodynamics, except for the reduction in PD (p < 0.001), with the largest difference being 13.8%. The differences in aVOR reduction were suggestive of statistical significance (p = 0.028), with the largest difference being 5.3% , EL (p < 0.001), and PD (p < 0.001), respectively, with the largest differences between treatments using first stents of different sizes being 6.9, 11.1, and 13.8%  and the IR   .r = 0.50), PD (r = 0.63), aVEL (r = 0.51), aVOR (r = 0.52), and aWSS (r = 0.53) . Reductions in the remaining haemodynamic parameters  were all very weakly correlated with changes in each of the three stent morphological parameters .The average pore size in stent wires spanning the aneurysm orifice is a morphological parameter that has been used less commonly than porosity or pore density in the haemodynamic analysis of flow-diversion treatments. Despite the fact that the three parameters are related to each other, we found that the average pore size was the only microstructural parameter demonstrating consistently good correlations with major haemodynamic parameters used in this study to assess the flow-diversion outcomes — e.g., the IR, PD, aVEL, aVOR, and aWSS , pore density (p = 0.002), and pore size (p = 0.003) were indicative of statistical significance  and 9 ± 12% (p = 0.056), relative to the untreated condition;deployment of a second stent further reduced the aneurysmal inflow rate (IR) and energy loss (EL) by 14 ± 11% ; andiii.r = 0.50, p < 0.001) and pressure drop .the change in stent pore size was the only microstructural parameter demonstrating a strong correlation with the reduction in the post-treatment aneurysmal haemodynamics, in terms of the IR (The size of the earlier-deployed stent has a significant impact on the IA haemodynamics after dual-stent treatment. The average pore size of stent wires at the aneurysm orifice shows promise as a potential index for predicting the efficacy of flow-diversion treatment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MZ and ST: guarantors of integrity of entire study, designed computational and analysis protocol, and wrote the manuscript. MZ: original concept. MZ, MO, and ST: collected patients’ data. MZ, ST, and YL: performed computational experiments. All authors analysed the data, revised manuscript for important intellectual content, approved final version of submitted manuscript, and agreed to ensure any questions related to the work are appropriately resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oryza sativa L.) is cultivated under a variety of climatic conditions. Due to the fragility of the coastal ecosystem, rice farming in these areas has lagged behind. Salinity coupled with floods has added to this trend. Hence, to prevent genetic erosion, conserving and characterizing the coastal rice, is the need of the hour. This work accessed the genetic variation and population structure among 2,242 rice accessions originating from India’s east coast comprising Andhra Pradesh, Orissa, and Tamil Nadu, using 36 SNP markers, and have generated a core set (247 accessions) as well as a mini-core set (30 accessions) of rice germplasm. All the 36 SNP loci were biallelic and 72 alleles found with average two alleles per locus. The genetic relatedness of the total collection was inferred using the un-rooted neighbor-joining tree, which grouped all the genotypes  into three major clusters. Two groups were obtained with a core set and three groups obtained with a mini core set. The mean PIC value of total collection was 0.24, and those of the core collection and mini core collection were 0.27 and 0.32, respectively. The mean heterozygosity and gene diversity of the overall collection were 0.07 and 0.29, respectively, and the core set and mini core set revealed 0.12 and 0.34, 0.20 and 0.40 values, respectively, representing 99% of distinctiveness in the core and mini core sets. Population structure analysis showed maximum population at K = 4 for total collection and core collection. Accessions were distributed according to their population structure confirmed by PCoA and AMOVA analysis. The identified small and diverse core set panel will be useful in allele mining for biotic and abiotic traits and managing the genetic diversity of the coastal rice collection. Validation of the 36-plex SNP assay was done by comparing the genetic diversity parameters across two different rice core collections, i.e., east coast and northeast rice collection. The same set of SNP markers was found very effective in deciphering diversity at different genetic parameters in both the collections; hence, these marker sets can be utilized for core development and diversity analysis studies.In India, rice ( Oryza sativa L.) is an important cereal crop which is a predominant food for over three billion people across the globe. Since it can adapt to an extensive spectrum of environmental conditions, it is therefore considered a varied crop species  are supposed to be the most bountiful variation found across the genome and therefore are excellent for high-resolution genotyping, in turn useful for analysis of genetic diversity and association mapping, linkage mapping, and marker-assisted selection (MAS) . Simple Plant Genetic Resources of native rice needs to be conserved promptly. A major conservation strategy counts on developing core collections, thereby screening gene bank accessions for important agronomic traits. To create a core collection, one needs a good construction strategy. In the present study, we demonstrate the identification of a diverse core set panel from the east coast region of India comprising 247 core accessions and a mini core set of 30 accessions to enable elite gene mining for breeding and conservation and on a long-term perspective to form a panel for association studies. We have also done a comparative analysis of genetic diversity parameters across two different rice collections , thus validating the use of SNP markers for studying diversity parameters across any other rice collection.A set of 2,242 seed samples of the east coast region of India  were procured from the Indian National Genebank, National Bureau of Plant Genetic Resources (NBPGR), New Delhi, with passport data  number) and the states to which they belong. This is given in www.sequenom.com). The information about the genetic location of the multiform assays designed for 36 SNPs having conserved single-copy rice genes was derived from The seed was de-husked, and genomic DNA was isolated using the QIAGEN DNeasy Plant Mini Kit. A tissue lyser  and a tissue lyser adapter set  was used to grind kernels into fine powder. Working stocks with 10 ng/μl of the genomic DNA samples was prepared, and 30 µl of the diluted sample was transferred to a 96-well plate to be run on the Sequenom MassARRAY which adopts the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer for most authentic detection of SNPs (http://taylor0.biology.ucla.edu). Additional hierarchical structure analysis was done after observing additional peaks at two different K values to unmask the groups   to calcue groups . Accessie groups  was usedPowerCore 1.0 software  was usedhttps://build.ngchm.net/NGCHM-web-builder, NG-CHM BUILDER: Interactive Heat Map  was used to determine the dissimilarity matrix which was used to construct an unrooted phylogenetic tree using FigTree v1.4.3 . Total cThe rice collections of coastal states belonging to the east coast of India were analyzed for the genetic diversity and population structure study. The lowest PIC recorded was 0.0 for marker 04-19-4_C from the state of Orissa, and the highest was 0.37 for marker v03-3478-1_C from the state of Andhra Pradesh. Heterozygosity was observed to be 0.0 for three markers 04-19-4_C, 11-3935_C, and 12-3200-2_C from Orissa state, and the highest value observed was 0.69 for marker 04-1801-20_C from the state of Andhra Pradesh. The lowest value of genetic diversity was found to be 0.0 for marker 04-19-4_C, and the highest was 0.49 for marker 02-267_C both from Orissa. The lowest and highest major allele frequencies recorded were 0.52 for 02-267_C and 1.0 for 04-19-4_C, respectively, from Orissa state . For thehttp://taylor0. biology.ucla.edu). The number of populations was found to be four for the entire 2,242 east coast rice collection . In the first case, four populations were assumed and AMOVA analyses revealed that 29% diversity exists among populations, 20% within individuals, and 51% among individuals of the total east coast rice collection , while tThe AMOVA study of Andhra Pradesh revealed 23% variance within individuals and 30% and 47% variance among populations and among individuals, respectively . PCoA plOut of 2,242 rice accessions studied, a core set of 247 accessions  and a mini core set of 30 accessions were selected using POWERCORE . Thirty-The degree of genetic diversity of the core set was studied to find the degree of genetic diversity captured from the overall coastal collection. Major allele frequency, gene diversity, heterozygosity, and PIC were observed to be 0.75, 0.34, 0.12, and 0.27 respectively . The valThe population structure grouped the core set accessions into four populations , 8. The The AMOVA study of the core collection revealed 29% variance within individuals as well as among population and 42% variance among individuals . The PCoKinship analysis of the core set showed that more than 50% of the samples had a kinship value less than zero, and less than 10% of the accessions had kinship values between 0.5 and 0.75 . The kin). This means that markers 01-608-4_C and 03-3478-1_C had given the maximum PIC value of 0.37 in both the collections and marker 04-19-4_C had given the minimum PIC value of 0.01 in both collections. Similarly, markers 01-608-4_C and 03-3478-1_C had displayed the highest gene diversity with a value of 0.49 across both collections and a minimum value of gene diversity 0.02 with marker 04-19-4_C across both collections. Generally, line graphs for PIC and gene diversity were overlapping for both collections except at certain points where deviations were observed. For example, marker 03-1691-1_C gave a gene diversity value of 0.32 in the coastal rice collection and 0.4 in the northeast rice collection. The same marker gave a PIC value of 0.26 in the coastal rice collection and 0.32 in the northeast rice collection. Marker 11-522-1_C gave a gene diversity value of 0.45 in the coastal collection and 0.19 across the northeast collection; also, this marker 11-522-1_C gave PIC values of 0.35 and 0.17 across the coastal rice and northeast rice collections. A subsequent analysis between major allele frequency and heterozygosity from the current study and with the northeast rice collection was also evaluated , markers 01-608-4_C and 03-3478-1_C were found to give the highest PIC value of 0.37, while marker 04-19-4_C was least informative giving the lowest PIC of 0.01 in both collections. Marker 01-608-4_C is a locus of evolutionary conserved genes from the saponin family of proteins, which is involved in the sphingolipid metabolic process and active in extracellular space ; in the Validation of the same set of SNP markers on two collections (northeast rice and east coast rice collection) has established that the 36-plex SNP assay is sufficient and efficient for initial diversity analysis and core development. Hence, this 36-plex SNP assay can be exploited by researchers for the genetic diversity study and development of core based on their own collections, thus accelerating their breeding program.This is the first study where India’s east coast rice collections were characterized using SNP markers. The genetic diversity and population structure were studied, and core and mini core collections with maximum diversity and minimum redundancy were developed. A total of 2,242 east coast rice accessions from three different states of India, i.e., Andhra Pradesh, Orissa, and Tamil Nadu, have been characterized, and a wide range of gene diversity and PIC was observed. A phylogenetic analysis of the total east coast rice collection revealed three groups, and a population structure analysis revealed four populations. The 36-SNP assay used in this study was validated by comparing the genetic diversity parameters  across two different rice collections, i.e., east coastal rice and northeast rice collection, and it was observed the these markers were sufficient to decipher all genetic parameters very efficiently; hence, they can be effectively utilized for core development and diversity study of different rice genotypes.


CA27.29 (MUC1) is a well described biomarker for prediction of prognosis and treatment efficacy. CA27.29 is mainly evaluated in the preoperative setting. However, testing of postoperative levels and additional assessment after chemotherapy might be more informative for analyzing the usefulness of CA27.29 in relation to the efficacy of chemotherapy. Thus, both pre- and post-chemotherapy values were assessed from patients enrolled in the breast cancer SUCCESS-A trial. Pre-chemotherapy assessment was associated with disease-free survival. It had no prognostic value in node-negative patients, but there was a clear association in node-positive patients. Furthermore, it was shown that post-chemotherapy CA27.29 assessment did not add any prognostic value, either on its own or in addition to pre-chemotherapy assessment. In conclusion, this indicates that pre- and post-chemotherapy values do not provide additional information. However, pre-chemotherapy CA27.29 could be a suitable tool to identify a group with unfavorable prognosis among node-positive patients.Soluble MUC1 has been discussed as a biomarker for predicting prognosis, treatment efficacy, and monitoring disease activity in breast cancer (BC) patients. Most studies in adjuvant settings have used preoperative assessment. This study, part of the SUCCESS-A trial (NCT02181101), assessed the prognostic value of soluble MUC1 before and after standard adjuvant chemotherapy. Patients with high-risk BC were treated within the SUCCESS-A trial with either three cycles of 5-fluorouracil, epirubicin, and cyclophosphamide followed by three cycles of docetaxel or three cycles of FEC followed by three cycles of docetaxel and gemcitabine. Cox regression analyses were performed to investigate the prognostic value of CA27.29 before and after chemotherapy relative to disease-free survival (DFS), along with established BC prognostic factors such as age, body mass index, tumor size, nodal status, estrogen receptor, progesterone receptor, HER2 status, and grading. Pre-chemotherapy and post-chemotherapy CA27.29 assessments were available for 2687 patients of 3754 randomized patients. Pre-chemotherapy CA27.29 assessment was associated with DFS in addition to established prognostic factors. It had no prognostic value in node-negative patients, but there was a clear association in node-positive patients. Post-chemotherapy CA27.29 assessment did not add any prognostic value, either on its own or in addition to pre-chemotherapy CA27.29 assessment. Biomarkers that help to monitor the efficacy of treatment may be extremely useful for predicting prognosis, guiding the therapy and assessing the early response to it . There hOne of these biomarkers is MUC1, which is overexpressed in many malignancies, particularly in breast cancer (BC) ,3. It coMUC1-N can be shed, is soluble in serum, and can be measured with antibodies directed against epitopes of this subunit. The shed and soluble subunit is often referred to using the name of the antigen (CA27.29 or CA15-3)—i.e., the target of the analytic assay for the measurement of MUC1 levels [MUC1 is a prognostic marker in both early ,16,17,18The aim of this study was therefore to assess the association with disease-free survival and MUC1 as measured using a CA27.29 assay before and after adjuvant chemotherapy, taking into account established prognostic factors.In the SUCCESS-A open-label phase 3 trial, patients were randomly assigned at a ratio of 1:1 to either an anthracycline/taxane-based chemotherapy or to this chemotherapy regimen plus gemcitabine . FurtherPatients were eligible if they were aged 18 or older and had a diagnosis of early, non-metastasized, high-risk invasive BC, defined by tumor stage, tumor grade, hormone receptor status or age. The full inclusion/exclusion criteria are provided in Additional Disease-free survival was defined as the time from the last chemotherapy administration to the earliest date of disease progression  or the date of censoring. Patients who were lost to follow-up before the maximum observation time of 5.5 years or were disease-free after the maximum observation time were censored at the last date on which they were known to be disease-free or at the maximum observation time. The maximum observation period of 5.5 years consists of six months of chemotherapy and (up to) five years of follow-up thereafter.For survival and recurrence assessment, the patients were followed at the study sites at 3-month intervals for the first three years and every six months thereafter. The follow-up included clinical examinations (at each visit), mammography (every six months), and symptom-driven examinations if necessary. High quality of the data was ensured by electronic data management, including automated plausibility checks and regular monitoring visits to the study site by an independent clinical research organization  and a data monitoring committee (DMC).g, room temperature) within 24–72 h following the collection time to remove clots. The serum was immediately transferred to an immunoreaction cup from the ST AIA-Pack 27.29 series  for further analysis. The CA27.29 serum concentration was measured using the AIA-600 II automated enzyme immunoassay system  in accordance with the manufacturer’s instructions. In brief, serum samples were combined with a diluent (1:20) and were transferred to an immunoreaction cup from the ST-AIA-Pack 27.29 series. CA27.29 was immobilized using magnetic beads conjugated to antibodies. Enzyme-labeled antibodies attached to a different epitope were then bound to the CA27.29 antigen in a sandwich manner. The samples were then incubated at 37 °C, followed by a washing step to remove any unbound antibody. The fluorogenic substrate 4-methylumbelliferyl phosphate was added to the test cup, and enzyme activity was measured on the basis of the amount of fluorescence.Laboratory analysis was performed centrally in the Department of Gynecology and Obstetrics at Munich University Hospital . ApproxiThe primary objective was to study whether information about CA27.29 before and after chemotherapy improves the ability to predict disease-free survival for each patient, in addition to other well-known predictors. For this purpose, Cox regression analyses were performed as described in the p value of <0.05 was regarded as statistically significant. Calculations were carried out using R statistical software . Remark criteria were used to report tumor marker data [All of the tests were two-sided, and a ker data .n = 2687). Complete information on all variables as listed in In total, 3754 patients were randomly assigned in the SUCCESS-A study. For inclusion in the present analysis, patients were required to have serum samples for CA27.29 analysis at the time of study inclusion and after chemotherapy . The effect of CA27.29 on survival differed between patient subgroups . The variable selection process resulted in a final Cox regression model that included, besides the predictors of the reduced model, the interactions of CA27.29 before chemotherapy relative to lymph node status (pN) and tumor size (pT). CA27.29 before chemotherapy was nonlinearly associated with survival, with two degrees of freedom, whereas CA27.29 after chemotherapy was best described as a linear predictor.CA27.29 was significantly associated with disease-free survival additionally to other predictors  improved with increasing CA27.29 values, but only up to values of 25 U/mL. Patients with CA27.29 values higher than 25 U/mL had a prognosis similar to that in patients with pT2 tumors—the higher the CA27.29 value, the poorer the prognosis. Moreover, the survival rates of patients with pT2 tumors were similar to those of patients with pT3/4 tumors in this range of above-average CA27.29 .No association between CA27.29 after chemotherapy and the disease-free survival was found. The adjusted HR per 10 U/mL increase of CA27.29 was 0.95 .In summary, a “high-risk patient” is a patient with a lymph-node positive tumor with at least pT2 and high CA27.29 values before chemotherapy or pT3/4 and low or high but not intermediate CA27.29 values before chemotherapy. The survival prognosis is further increased or decreased, respectively, by histology, grading, ER, PR, and HER status. For instance, a patient with a tumor classified as T2, G3, ER+, PR+, and HER− is predicted to have a 5-year disease-free survival with a likelihood of 85% if the preoperative CA27.29 level was low and a likelihood of 75% if the preoperative CA27.29 was high. Both the full and final models performed better than the basic model without CA27.29 predictors with regard to distinguishing between patients with and without events up to two and five years of follow-up, respectively . In the latter, low CA27.29 values were associated with an unfavorable prognosis. Interactions between molecular subtypes were not analyzed since the assumptions for the proportional hazard function were not met and there was a strong correlation between ER and PR. Several larger studies have reported associations between tumor size, nodal status, and MUC1 ,17. PatiOnly one study has investigated the association of postoperative MUC1 assessments in relation to other prognostic factors  . CarcinoMUC1 has been widely discussed as a prognostic biomarker and potential target for anticancer treatment ,45. In aMUC1 has been prioritized as a promising target for anticancer therapies ,45. VaccThis study has several strengths and weaknesses. It is the first large-scale study in which samples have been analyzed in a multicenter prospective phase III chemotherapy investigation. CA27.29 was assessed centrally, ensuring high-quality data and standardized analytic methods. It therefore appears to be justifiable to generalize the results, although the study was restricted to a high-risk group undergoing chemotherapy. While the overall sample size was reasonably high with more than 2600 patients, the unfavorable prognostic effect in patients with pT3/4 and low CA27.29 may be imprecise due the small sample size for that specific patient group.In conclusion, this study did not show additional prognostic value for serial CA27.29 assessment before and after chemotherapy alongside commonly used prognostic factors and postoperative CA27.29 measurements. In combination with tumor size and nodal status, as well as other prognostic factors, however, serial CA27.29 measurements can identify patients with an unfavorable prognosis. This patient population may be suitable for further anti-MUC1 treatment after standard therapies.